ResearchThe general focus of my research revolves around the study of the coordination of tissue specific gene expression during differentiation, from the cell surface to changes in gene transcription. To understand this process we have taken a systems level approach utilizing microarray and proteomic methodologies in conjunction with inhibitor and promoter analysis of MC3T3-E1 cells. My studies are currently focused on the role of inorganic phosphate that is generated during osteoblast differentiation, as a novel-signaling molecule capable of ultimately altering gene expression of a number of phosphate responsive genes including the transformation and metastasis associated osteopontin, as well as the Nrf2, a key transcription factor in the response of phase I detoxifying enzymes and putative target for chemoprevention, The study of this model system and the regulation of these genes in particular provide the potential that future results might impact a number of disease states including cancer, bone metabolic disorders, osteoarthritis, atherosclerosis and kidney disease.A systems level approach to understanding inorganic phosphate signaling and the regulation of Osteopontin and Nrf2 Our previously published results detailed the discovery that elevated inorganic phosphate results in increased expression of osteopontin not only in MC3T3-E1 cells but also in other cell types including the fibroblast cell line NIH3T3, defines a novel mechanism for gene regulation. Osteopontin is a multi-faceted extracellular matrix protein expressed in most tissues. Studies involving reduced levels of osteopontin have shown that it is required for bone remodeling, wound healing and as a key cytokine in efficient type-1 immune responses. Elevated levels of the osteopontin are associated with various disease states including transformation, metastasis, atherosclerosis and kidney disease. A high level of expression of osteopontin is associated with the poor prognosis of many cancers. Although expression of this gene has been demonstrated in multiple tissue types its regulation is not well understood. We have identified three key signaling pathways in osteopontin expression including ERK1/2, PKC and the proteosome. The proteasome is required for expression of osteopontin not only in response to phosphate but also in response to the tumor promoters, TPA and okadaic acid. This suggests the proteasome may be an important component in the regulation of osteopontin not only in osteoblasts in response to elevated phosphate but in the global regulation of osteopontin expression in response to a host of stimuli. Furthermore, given the correlation between elevated osteopontin RNA levels and the metastatic phenotype of many cancer cells these results may provide insight into evaluating proteasome inhibitors as cancer therapeutics. We are currently defining promoter element(s) and transcription factor(s) that regulate osteopontin expression in response to elevated phosphate. Using Microarray technology we have determined that, in fact, numerous genes are regulated by elevated inorganic phosphate including, Nrf2 a key transcription factor in the regulation of phase II detoxifying enzymes, responsible for the detoxification of carcinogens. Loss of function of Nrf2 has been linked to carcinogenesis. We have identified Nrf2 as an immediate response gene to phosphate stimulation during osteoblast differentiation, and cloning of the promoter confirmed that Nrf2 is transcriptionally regulated by increased inorganic phosphate. We continue to study the Nrf2 promoter with the goal of understanding how this key factor in chemoprevention is regulated.The goal of fully understanding the cellular response to a given stimuli or changing environment is currently limited by the inability to fully evaluate the proteome. The ability to conduct an analysis of changes in both protein abundance and RNA levels on a systems scale sufficient to entirely enumerate the response of a cell will undoubtedly be a powerful tool in assessing not only complex cellular processes such as differentiation but should also provide clues as to how to manipulate the cell phenotype by intervention. To this end we haveconducted a coordinated proteome and microarray analysis of inorganic phosphate treated pre-osteoblasts. The results from these studies describe one of the largest proteome databases derived from differentially treated mammalian samples. A comparison of the cICAT derived protein data with microarray samples has lead to the determination that analyzing RNA levels at numerous time points leading up to a given proteome time point may give a more accurate picture of the transcription/translation relationship. Furthermore this type of comparison can lead to novel methods of data extraction including the identification of posttranslationally regulated proteins as evidenced by Fra-1. The data presented here reveal not only novel insights into the role of inorganic phosphate in altering cell function but also provide an initial glimpse at the power of combining large scale protein and RNA analysis for a systems level understanding of the cell. The swi/snf complex and adipogenesis.A related project in the lab revolves around the role of Smarcf1 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily f, member 1) in differentiation. Smarcf1 is an integral member of human SWI/SNF complexes. These complexes are chromatin modifying machines that play fundamental roles in the regulation of gene expression during cell growth and development in all organisms. Sequence analysis revealed that Smarcf1 has a group of four LXXLL (L is leucine and X is any amino acid) motifs located towards the C-terminus of the protein. This motif has been demonstrated to be necessary and sufficient for interaction with liganded nuclear hormone receptors. The function of other LXXLL containing proteins is to assist in the formation of a complex between upstream transcriptional activators/enhancers and the basal transcriptional machinery, including TBP. The potential interaction between various nuclear hormone receptors and Smarcf1, a protein associated with a complex that contains chromatin-modifying activity suggests a role for Smarcf1 as an integrator of hormone signaling and transcription initiation. We are currently using a model of adipocyte differentiation to examine the potential role(s) of Smarcf1 and the SWI/SNF complex in differentiation.